Campylobacter fetus is a significant pathogen of cattle and immunocompromised humans. One of the major virulence factors of C. fetus is its surface layer (S-layer), which allows it to resist the bactericidal effects of normal and immune serum. Despite significant advances in understanding the molecular mechanisms of antigenic variation of surface layer proteins (SLPs, the subunits of the S-layer), until recently little was known about the secretion and assembly of SLPs. Preliminary data now reveal that C. fetus SLPs are transported by a type I secretion system [Sap(C)DEF] similar to those that recognize C-terminal secretion signals for the transport of toxins, proteases and lipases from gram-negative bacteria. This relationship is demonstrated by the inability of a C. fetus sapD mutant to produce or secrete SLPs, suggesting a possible link between the secretion and synthesis of SLPs. Furthermore, E. coli expressing C. fetus sapCDEF are able to specifically secrete a C. fetus SLP (SapA), verifying the sufficiency of these genes for SLP secretion. The ability of sapCDEF+ E. coli to secrete SLPs will be exploited for the delineation of the SapA C-terminal secretion signal. The investigators therefore propose the study of the mechanism of secretion of C. fetus SLPs as a necessary component of the process by which a major virulence factor of C. fetus is assembled, and as a model system for examining the interactions between heterologous type I transporters. They will do so in the following specific aims. Specific Aim #1. To characterize the components of the SLP secretion apparatus of C. fetus through the construction of additional mutations in the secretion apparatus. Studies on the potential regulation of sapA expression by intracellular SLPs will be performed to investigate a possible link between SLP synthesis and secretion. Specific Aim #2. To construct a C. fetus SLP secretion assay system using the cloned sapCDEF genes in E. coli. They will use deletion and mutational analyses to define the C. fetus SapA C-terminal secretion signal. The ability of the SapA secretion to mediate the transport of normally non-secreted proteins also will be assessed.